Project Summary/Abstract The purpose of the research program is to discover new anti-HIV drugs that are potent, safe, easily administered, and long-acting. The approach combines state-of-the-art technology for molecular design, synthetic organic chemistry, biological assaying, and crystallographic determination of structures of the designed molecules bound to their protein target. The PI's research program emphasizes fundamental advances in the development of software and methodology for drug design, detailed modeling of protein-ligand binding, and organic synthesis. The PI's group has developed computational tools to speed lead optimization for potency, while being mindful of the need for desirable pharmacological properties. For the HIV work, collaborations with scientist in the Yale School of Medicine provide the determinations of biological activity in vitro, in human T-cells, and in humanized mouse models, as well as macromolecular structures through protein crystallography. The specific focus is the discovery of inhibitors of HIV-1 reverse transcriptase (HIV-RT), which are a central component of highly active antiretroviral therapy (HAART). The previous grant period witnessed striking advances for our development of the catechol diether series of non-nucleoside inhibitors of HIV-RT. Compounds have been discovered that show remarkable potency, no cytotoxicity, no off-target activity, excellent pharmacological properties, and efficacy in a humanized mouse model of HIV-1 infection. From our extensive crystallographic and modeling studies, thorough knowledge of the binding site has also led to an important new direction of developing the first covalent inhibitors of HIV-RT (CRTIs). We initially targeted the clinically problematic Tyr181Cys RT variants and designed CRTIs that completely knock out activity of the resistant mutants. Conclusive evidence for the covalent modification of Cys181 is provided from enzyme inhibition kinetics, mass spectrometry, protein crystallography, and antiviral activity in infected human T-cell assays. The CRTIs were also shown to be selective for Cys181 and have lower cytotoxicity than the approved drugs efavirenz and rilpivirine. Our compounds are rare examples of targeted covalent allosteric inhibitors, which have enhanced potential for low dosage, low toxicity, and extended duration of action. Extensive discovery, characterization, and development of CRTIs targeting both wild type and resistant forms of HIV-1 are the focus for the next grant period. CRTIs are a new class of anti-HIV agents with potentially profound therapeutic impact.